Project Summary Recent studies have identified intestinal microbe-derived metabolites such as Trimethylamine-N-oxide (TMAO) as a novel risk factor for cardiovascular diseases (CVDs). TMAO, a gut microbe-derived metabolite of dietary phosphatidylcholine/carnitine is elevated in the circulation of CVD patients and has been associated with atherosclerosis and CVD progression in rodents and humans. In spite of this striking association, the molecular mechanisms of how TMAO induces atherosclerosis and CVD progression are still unclear. In this grant proposal, we attempt to elucidate an early intracellular molecular mechanism, namely, the Nlrp3 inflammasome activation, which may switch on endothelial damage through its inflammatory or non-inflammatory pathway leading to endothelial dysfunction and ultimately atherosclerosis. Interestingly, our preliminary studies demonstrated that TMAO-induces the Nlrp3 inflammasome activation and contributes to the endothelial damage and microvascular injury and have also shown that beyond inflammation, the activated inflammasomes have direct actions on the endothelial cells. This may represent a novel pathogenic mechanism of inflammasome activation beyond inflammation. Based on these observations, we hypothesize that gut microbial metabolites such as TMAO which are released into the circulation act as endogenous danger signals and induce both inflammatory and non-inflammatory responses via Nlrp3 inflammasome activation leading to endothelial dysfunction and vascular injury which consequently manifests into atherogenesis in the arterial wall. To test this hypothesis, we will first determine whether TMAO-induced Nlrp3 inflammasome activation contributes to tight junction disruption, altered vascular permeability, endothelial dysfunction and atherosclerosis in vivo using Nlrp3-/- mice, endothelium-specific Nlrp3 knockout mice (EC-Nlrp3-/-) and their wild type littermates. We will then study how Nlrp3 inflammasomes are activated in endothelial cells by TMAO with a focus on the roles of NADPH oxidase mediated redox signaling and corresponding mechanisms mediating its actions. Finally we will determine the non-inflammatory and inflammatory effects of TMAO activated Nlrp3 inflammasomes on endothelial dysfunction and atherosclerosis by studying the various products such as IL-1?, IL-18, pyroptosis and DAMPs in primary cultures of CAECs and carotid arteries of Nlrp3-/- and Nlrp3+/+ mice. The proposed studies will reveal new mechanistic insights of CVD pathogenesis induced by microbial metabolites such as TMAO and will pave way to the development of clinically relevant, novel therapeutic strategies for treating atherosclerosis and other cardiovascular disorders.